TEST AND MODEL FOR POLYCYSTIC OVARY SYNDROME AND MALE PATTERN BALDNESS

Abstract: Polycystic ovary (PCO) syndrome has been discovered to have an underlying genetic cause; further, in males the phenotype of the disorder has been found to be male pattern baldness (MPB). A mutation in the CYP17 gene, which encodes the enzyme P450c17, demonstrates an association with the phenotype: the mutation is a replacement of a T by a C within the 5' transcribed region of the gene. Recombinant DNA systems for modelling, diagnosing and treating the disease are based on these findings.

Description: TEST AND MODEL FOR POLYCYSTIC OVARY SYNDROME AND MALE PATTERN BALDNESS This invention is based on the identification of a genetic cause of polycystic ovary syndrome and male pattern baldness and relates to the application of that discovery for diagnostic, therapeutic and modelling purposes. Polycystic ovary syndrome (PCOS) is a very common endocrine disorder (Adams et al , The Lancet (ii) :1375-78 (1985), Adams et al, Bri tish Medical Journal 293:355-59 (1986) , Fox et al , Clinical Endocrinology 34:127-131 (1990), Hull, M.G., Gynecol . Endocrinol . l(3):235-45 (1987) , Sagle et al, British Medical Journal 297(6655) :1027-8 (1988) and Poison et al, The Lancet 1(8590) :870-2 (1988)) , the aetiology of which is unclear. It is clinically heterogeneous, presenting with one or a combination of symptoms, including hirsutism, menstrual disturbances or infertility. This has led to scepticism as to whether there is a single underlying abnormality. The most consistent biochemical finding in all groups of women with PCO, regardless of clinical presentation, is an elevation of serum androgens (Franks S., Clin. Endocrinol . Oxf . 31(1):87-120 (1989) and Franks S., Journal of Endocrinology 129:317-319 (1991)). This supports the hypothesis that, despite the variable clinical phenotype, polycystic ovaries are caused by a common underlying disorder of androgen biosynthesis or metabolism. Several reports have observed familial clustering of cases of PCOS (Goldzicher et al, Journal of Clinical Endocrinology 22:325 (1962) , Llague et al , Clin . Endocrinol . Oxf . 29 (6) :593-605 (1988), Cooper et al , American Journal of Obstetrics and Gynaecology 100:371-87 (1968) , Givens J.R., Endocrinol . Metab . Clin . North . Am . 17(4) 771-83 (1988), Ferriman et al , Clinical Endocrinology 11:291-300 (1979) and Lunde et al, Gynecol . Obstet . Invest . 28(l) :23-30 (1989)), suggesting a genetic component, but none has been able to demonstrate a clear mode of inheritance. A dominant mode of inheritance has been suggested, based on family histories obtained from patients (Ferriman et al , Clinical Endocrinology 11:291- 300 (1979) ) but in a recent study in which affected relatives were identified by ovarian ultrasonography, the prevalence of PCO in the families was too high to be explained by simple Mendelian dominant inheritance (Llague et al , Clin . Endocrinol . Oxf . 29 (6) :593-605 (1988) ) . In a recent review of the available literature, Simpson concluded that the mode of inheritance remained unclear and that heredity due to a single gene defect seemed unlikely (Simpson J.L., Elucidating the genetics cf polycystic ovary syndrome. In: Dunaif A., Givens J.R., Haseltine F.P., Merriam G.R., eds. Polycystic Ovary Syndrome. Oxford: Ξlackwell Scientific Publications, 1992: 59-77) . There are, however, a number of problems in interpreting previous studies which include varying diagnostic criteria for PCOS. Critically, many of the larger studies have used proband interviews or questionnaires as the sole source of information about first degree relatives and extended family members; none has included interviews and examination of male family members. In addition, there have been few attempts to exclude late- onset (non-classical) congenital adrenal hyperplasia and the ethnic origin of the patients is rarely mentioned. Little attention has been paid to a possible male phenotype, although two studies have shown an association between PCOS and premature male balding (male pattern baldness; MPB) (Ferriman et al , Clinical Endocrinology 11:291-300 (1979) and Lunde et al , Gynecol . Obstet . Invest . 28(l) :23-30 (1989)) . In studies underlying the present invention, the mode of inheritance for ten large families has been determined by identification through affected members and classical segregation analysis. It has been discovered chat PCO has an underlying genetic cause and that the male phenotype of the disorder is male pattern baldness (MPB) . It has also been discovered that a polymorphism in the gene CYP17 demonstrates an association with the disease. The association of the mutation with PCO and/cr MPB is short of complete segregation, which suggests that CYP17 is one of the major modulating genes for polycystic ovaries and male pattern baldness, but is not causative alone. The CYP17 gene, also designated P450XVIIA1, lies on chromosome 10 and encodes P450cl7, which is a single cytochrome P450 enzyme mediating both 17α-hydroxylase and 12,20 lyase activities in the biosynthesis of steroid hormones. The gene was cloned and sequenced by Picado- Leonard & Miller (DNA 6(5) 439-448 (1987)), whose numbering system, as laid out in Figure 3 of their paper, is adopted in this specification. ?450cl7 acts as a 17α.-hydroxyiase on pregnenolone and progesterone to form 170H-pregnenolone and 170H- progesterone, respectively. It further acts, as a 17,20 lyase, en 170H-pregnenolone and 170H-progesterone to form DHEA and androstenedione. P450cl7 is believed to be the rate-limiting enzyme in androgen biosynthesis. The invention generally provides model systems of polycystic ovary syndrome (PCO) and/or male pattern baldness (MPB) and DNA molecules which are useful in such model systems and/or in diagnosis or genetic therapy; the model systems and DNA molecules comprise a DNA sequence including at least part of the CYP17 gene which includes a mutation leading to increased androgen biosynthesis. According to a first aspect of the invention, there is provided an isolated cr recombinant DNA encoding a mutant CYP17 allele which is associated with polycystic ovary syndrome (PCO) and/or male pattern baldness (MPB) . The mutation may be in the protein-coding region of the CYP17 gene and may be responsible for increased P450cl7 enzymic activity. Alternatively, the mutation may be in the promoter region, which is untranslated and lies 5' to the protein-coding region. Such mutations may lead to increased expression of the gene product. In a particular embodiment, the DNA sequence includes at least part of the 5' untranslated region of the CYP17 gene in which T is replaced by C at position -34 from the translation initiation site. The T→C change results in the generation of an SP-1 type site and is believed to up-regulate the gene by this means. Consequently, the mutation results in increased androgen biosynthesis. The DNA sequence may include the entire functional promoter, in some embodiments preferably with the protein-coding region. cDNAs and genomic DNAs are encompassed within the scope of the invention. A particular embodiment of the invention relates to an isolated or recombinant DNA including a mutant CYP17 allele having a T→C mutation at position -34. Recombinant DNA in accordance with the invention may include a mutant CYP17 promoter operatively coupled to a gene (a "reporter gene") to which the promoter is not normally operatively coupled and which encodes a detectable protein (a "reporter") , which may be an enzyme catalysing a detectable change, for the purpose of studying the promoter. Recombinant DNA of the invention may be in the form of a vector, such as a plasmid, cosmid, phagemid or virus, which may be used to transform or transfect a host cell. Vectors including a promoter operatively coupled to protein-coding DNA may be useful as expression vectors; vectors unable to express protein-encoding DNA may still be useful as cloning vectors. According to a second aspect of the invention, there is provided a host cell transformed or transfected with a vector as described above. The host cell may be prokaryotic or eukaryotic. A suitable prokaryotic host may be Escherichia coli . Suitable eukaryotic hosts include yeast (such as Saccharomyces cereviεiae) , insect cells (such as Ξpodoptera frugiperda) and mammalian cells, which may be primary cells or an immortalised cell line. Recombinant or isolated DNA in accordance with the invention may be in the form of a construct suitable for generating a transgenic animal. According to a third aspect of the invention, there is provided a transgenic, non-human animal whose germline includes such a construct, present as a transgene. Transgenic animals of the invention therefore include those having a transgene encoding a mutant CYP17 allele which is associated with PCO and/or MPB. Such transgenic animals may be of any desired species and may be prepared by methods known in the art. For mice, the standard Hogan microinjection protocol (Hogan et al., "Manipulating the Mouse Embryo: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA (1986)) may be used. Transformed or transfected cells and transgenic animals in accordance with the invention are useful not only for studying the genetic basis of PCO/MPB and its associated biochemistry but also for screening for drugs and evaluating drug effectiveness. According to a fourth aspect of the present invention, there is provided the use of such a cell or animal in a screen for a compound potentially useful in the treatment or prophylaxis of PCO and/or MPB. A compound under test may be provided or administered to the cell or animal; androgen biosynthesis, particularly as mediated by ?450cl7, may be monitored. The invention also has application in determining a genetic predisposition to PCO and/or MPB and other genetic analysis. According to a fifth aspect of the invention, there is provided a method of genetic analysis, particularly for the purpose of . determining a genetic predisposition of a subject to PCO and/or MPB, the method comprising detecting in the subject's DNA the presence of a CYP17 allele. Detection, which may involve DNA sequencing, may be carried out on material removed from, and not returned to, the subject's body. Detection may alternatively or additionally involve the use of probes, in which case oligo- or poly-nucleotides as described below may be useful. According to a sixth aspect of the invention, there is provided a oligo- or poly-nucleotide specifically hybridising to a mutant CYP17 allele having a T→C mutation at position -34. The hybridisation conditions will be sufficiently stringent to result in specific hybridisation. The oligo- or poly-nucleotide, which may be at least 10, 15, 20 or 25 nucleotides in length, in increasing order of preference, may be labelled with a detectable label, such as a radioisotope. However, that is not essential. For example, the oligo- or polynucleotide may be used as a primer in a polymerase chain reaction-based detection method. The fact that the T→C mutation at position -34 results in a Msp l restriction site means that, for this specific mutation at least, the PCR reaction product may be analysed by restriction enzyme digestion. Such a detection method could also be used in a PCR-based method which used primers based on sequences nearby the site of the mutation, rather than taken from the mutation site itself. Having detected a genetic mutation in the CYP17 gene in an individual it may be possible to employ gene therapy, in the form of gene implants, to prevent the development of PCO/MPB. A variety of ways may be used to restore normal, or at least acceptable, levels of CYP17 expression. For example, a normal CYP17 gene or promoter may form the basis of a gene implant; alternatively, the production of P450cl7 by a mutant CYP17 gene, or the activity of the P450cl7 so produced, may be modulated. Embodiments directed to modulation of the production of P450cl7 include methods that employ specific antisense polynucleotides complementary to all or part of a variant CYP17 gene or, more specifically, promoter. Such complementary antisense polynucleotides may include nucleotide substitutions, additions, deletions or transpositions, so long as specific hybridisation to the relevant target sequence, which in preferred embodiments will be in the region of position -34, is retained as a property of the polynucleotide. An antisense polynucleotide should preferentially bind to a variant CYP17 gene sequence as opposed to wild type CYP17. Preferred features of each aspect of the invention are as for each other aspect, mutatis mutandis. In work culminating in the invention, the families of patients identified sequentially as having polycystic ovaries (PCO) were examined in detail to determine their genetic structure, ovarian morphology, clinical and endocrine status. Ten families were found to have sufficient members for further study. First degree female relatives of affected individuals had a 51% chance of being affected. Early onset male pattern baldness (MPB) was found to be an accurate phenotype for obligate male carriers. All families showed autosomal dominant inheritance for PCO with greater than 90% penetrance. The frequency of the autosomal PCO in the population is approximately 20%. It appears that PCO and male pattern baldness are caused by alleies of the same gene, which affect androgen production or action. The different frequencies of PCO and MPB arise from differing thresholds for phenotypic expression in females and males respectively. The modifying effects of other genes is the most likely explanation of the somewhat variable phenotype. The invention will now be illustrated by the following non-limiting examples. EXAMPLE 1 Evidence for a Single Gene Effect causing Polycystic Ovary Syndrome and Male Pattern Baldness Patients were recruited prospectively and consecutively from the endocrine and infertility clinics at the Samaritan and St. Mary's Hospitals, London (probands) . Each proband had been diagnosed as having polycystic ovaries (PCO) by pelvic ultrasound scan and was known to have at least one sister, whether or not she was thought to be affected. The ultrasound scan was performed using an ACUSON 128 ultrasound machine and the ovarian morphology defined as previously described (Adams et al , The Lancet (ii):1375-78 (1985), Adams et al, Bri tish Medical Journal 293:355-59 (1986)) . The probands were of several racial and national origins which were noted, and presented with a variety of symptoms. Nine of the 14 women had irregular menses (four of these were also hirsute) , four presented with hirsutis but regular cycles and one had a history of primary infertility. Each was interviewed and a full history was taken, with particular reference to criteria for menstrual disturbance as described by Poison et al (Poison et al , The Lancet 1(8590) :870-2 (1988)), and subjective assessment by the subject of the degree of acne and hirsutism. A general examination was performed including height, weight and blood pressure measurements. The degree of any hirsutism was assessed using the Ferriman and Gallwey score (Ferriman et al , Journal of Clinical Metabolism 21:1440-5 (1961)) and the presence or absence of acne noted. Body mass index (BMI) was calculated for each subject, although obesity was not taken as a specific symptom of PCOS. Similarly, a history of recurrent miscarriage and infertility was noted, but not used as a positive predictor. A blood sample was taken for gonadotrophins (FSH and LH) , testosterone, prolactin and 17-α-hydroxyprogesterone estimations by radioimmunoassay (Adams et al, The Lancet (ii) :1375-78 (1985) and Franks S., Clin . Endocrinol . Oxf . 31(1) :87-120 (1989) . The presence or absence of acanthosis nigricans and other clinical features of type A insulin-resistance (Moller et al, The New England Journal of Medicine 325:938-948 (1991)) was noted in all family members. The glucose and insulin response to a standard 75g oral glucose tolerance test was assessed in obese probands. All first degree relatives of 14 probands were contacted, from which ten kindreds were identified with a family .structure which could be analysed and with members who were available for sampling and assessment for this study. Each first degree relative in these ten kindreds (62 individuals) was interviewed and screened according to the above protocol. In addition, each male family member was also assessed for the degree and time of onset cf balding. Premature male baldness has been defined as significant fronto-parietal hair loss before the age of 40 (Ferriman et al, Clinical Endocrinology 11:291-300 (1979) ) , but for the purposes of this study we took 30 years as the lower age limit. A further 107 extended family members were also contacted and interviewed and data were obtained for the 24 deceased members of these families ' from other relatives. Statistical analysis of frequency differences between groups was evaluated using a % *** or Fisher's exact test. Student's t-test was used for calculating differences between means in endocrine and ultrasound measurements. Statistical significance is taken as P<0.05. Approval for this study was obtained from the ethics committee of Parkside Area Health Authority. A total of 81 kindred members of whom only one is dead, were identified as first degree relatives of the 14 probands recruited into this study. Full screening was possible for 69 members of these pedigrees and interviews were conducted with a further six members. Five members cf one family lived abroad and could not be contacted. Of the 11 families, ten were Caucasian (two Irish), two Iranian, one Afro-Caribbean and one Asian. Four families were excluded (two Caucasian, one Afro-Caribbean and one Iranian) from the segregation analysis as it was not possible to complete the screening for all first degree relatives. From the ten kindreds, 58 of 62 members were fully screened, one was deceased and three (two male and one female sib) declined full screening, but were available for interview. There were also 88 known, extended family members from these ten kindreds; full screening of 28 of these (27 from two large kindreds) has been possible to date. The medical histories were also obtained of the 24 deceased members. A total of 89 from all the kindreds were completely screened, 67 women and 22 men. Of these women 17 were excluded from statistical analysis; three were premenarchal; nine postmenopausal and one was pregnant. In three, the ovarian morphology could not be reliably assessed and one woman declined to have the ultrasound scan. Four women had blood samples taken at mid-cycle and their LH values were excluded from this analysis. A history was taken to be positive for PCOS if it included menstrual irregularity (prior to commencing a combined oral contraceptive pill) and/or unwanted hair, with or without acne. The results of 17α-hydroxyprogesterone and prolactin estimations in these kindreds were all within the normal ranges. No families showed the typical phenotype of type A insulin resistance. There were eight probands with a BMI>25. In none of those tested was the insulin response to oral glucose outside the range for the obese control group. Of the 50 women available for analysis 16 were taking a combined oral contraceptive pill (COCP) . Where necessary (eg for LH and testosterone measurements) the results of these two groups have been considered separately. Eleven of the 16 taking a COCP at the time of screening were using it for treatment of menstrual disturbance or hirsutism. As expected, the mean [SD] ovarian volume were greater in those women with a polycystic morphology on ultrasound than in those with normal ovaries (PCO: 13.1 [4.9] cm 3 , normal: 4.6 [1.7] cm 3 ). Despite the fact that 15 women were taking an oral contraceptive pill 29 of the 37 women (78%) with PCO had an ovarian volume >9ml 3 , two standard deviations from the mean for normal ovaries (Poison et al , The Lancet 1(8590) :870-2 (1988)). Of the women taking the COCP 15 of 16 had PCO. There was a highly significant association of ovarian morphology with a positive history of PCOS (p<0.0034); 84% of women having an ultrasound diagnosis of PCO were symptomatic. Thirty one of the 32 women with irregular menses and/or hirsutism had PCO (97% sensitivity) ; 31 of 37 women with PCO were symptomatic (a positive predictive value of 84%) . Conversely a negative history had a 92% negative predictive value. There was no correlation between ovarian morphology or symptomatology and BMI. For those who are not taking oral contraceptives, the LH values (mean [S.D.]) for normal women differed significantly from those for women with PCO (normal: 5.0 [2.4] U/I, PCO: 10.2 [4.6] U/I; p=0.003). When those taking oral contraceptives are included, there is no significant difference in the LH values. Serum testosterone concentrations were significantly higher (p=0.015) in women with PCO (2.2 [0.7] nmol/1) than in those with normal ovaries (1.5 [0.5] nmol/1). This difference was sustained if the data on those women taking the oral contraceptive were also included (p=0.007) . Using biochemical abnormalities as indicators of PCO it was found that combining LH and/or testosterone measurements identified 54% of women that had PCO on ultrasound. A combination of symptomatology with biochemical markers was able to identify 92% of women with ultrasound evidence of polycystic ovaries (p<0.001) . Of the 22 men screened in this study, eight demonstrated premature male pattern baldness, ten were normal and four were too young to be evaluated. Two of the normal subjects were excluded from endocrine analysis, one was taking high dose steroids after a renal transplant and the other had a very low serum testosterone concentration (4.4nmol/l) and is currently under further investigation. There was no association of BMI with premature MPB. The mean testosterone levels were slightly, but not significantly, greater in the men with MPB compared to non-affected men (12.3 [4.2] nmol/1, vs 10.7 [4.2] nmol/1) . The prevalence of premature MPB in these families (31%) is in agreement with that reported in previously published studies (Ferriman et al , Clinical Endocrinology 11:291-300 (1979) and Lunde et al, Gynecol . Obstet . Invest . 28(l):23-30 (1989)) and much greater (χ 2 =19.3; p=0.0001) than the prevalence in families known not to have PCO (7%) (Ferriman et al, Clinical Endocrinology 11:291-300 (1979) and Lunde et al, Gynecol . Obstet . Invest . 28(l):23-30 (1989)). Assignment of affection status was then determined for each member of the ten pedigrees on the basis of the screening procedure. Affected status was allocated, on the basis of a positive history, to post menopausal women and those who had undergone a surgical menopause. The men were assigned a positive affection status on the basis of the history of premature MPB. This also allowed assignment of 12 deceased males using histories obtained from close family relatives which, in eight cases, was supported by photographic evidence. In family N.S. it has not been possible to assign the female member II 4 because she is postmenopausal and was not able to provide a reliable gynaecological history. The female members 111-10 and 11 are not assignable as they are premenarchal. The segregation ratio calculated for the affected families, excluding the proband to avoid ascertainment bias, was 51.4%. In every case, if male pattern baldness is taken as the male phenotype, the segregation is consistent with autosomal dominant inheritance. In this study it has been demonstrated that the typical findings of PCO on ultrasound have a high concordance with a symptomatic history (84%) . When symptoms were combined with a biochemical marker we were able to diagnose 92% of affected individuals. These data are in agreement with a previous study from this centre in which the prevalence of PCO was assessed in a normal population (Poison et al, The Lancet 1(8590) :870-2 (1988)) . In that study 94% of women with PCO could be identified by symptoms alone. These observations are important in determining a mode of inheritance for PCO, because to date, the ability to assign affection status has been limited to women of reproductive age, thus reducing the analysis to one or, at best, two generations. Since a positive history is such a strong predictor for polycystic ovaries,it would seem reasonable to assign women for whom full screening is not possible and from whom a reliable history can be obtained, an affection status on this basis alone. This study has found that ovarian volume, LH and testosterone values for the normal and PCO groups, whether or not they are taking an oral contraceptive, are consistent with the results of previous studies (Poison et al , The Lancet 1(8590) :870-2 (1988) and Franks S., Clin . Endocrinol . Oxf . 31(1) :87-120 (1989)) . The prevalence of premature MPB in the families studied was higher than expected for the non-affected families, as has been reported previously (Ferriman et al , Clinical Endocrinology 11:291-300 (1979) and Lunde et al, Gynecol . Ojbstet. Invest. 28(l):23-30 (1989)). When the assignment of affection status was made, presuming premature MPB as the male phenotype, positive ultrasound for postmenarchal premenopausal women and a positive history for postmenopausal women, the segregation ratio, excluding the index case, is 51.4%. For all ten families the mode of inheritance was consistent with an autosomal dominant disorder. Furthermore, this model was appropriate in three of the other four families where all the first degree members were accessible but affection status was assigned on interview alone. In both this and a previous study (Poison et al, The Lancet 1 (8590) :870-2 (1988)) it has been demonstrated that 8% of normal women who have ultrasound evidence of PCO do not have menstrual disturbances and or hirsutism, although a more recent population study suggests that a rather higher percentage of women with PCO are asymptomatic (Clayton et al, Clinical Endocrinology 37:127-134 (1992)) . It is conceivable that some of these women may present with recurrent early miscarriage. It is known that approximately 80% of these women have polycystic ovarian morphology of which less than 40% have any other symptoms (Sagle et al , Bri tish Medical Journal 297(6655) :1027-8 (1988) ) . What is the underlying abnormality in this disorder? An increased mean serum testosterone concentration is characteristic of each group of women with PCO, including those with regular cycles who present with hirsutism or history of recurrent miscarriage (Sagle et al, Bri tish Medical Journal 297 (6655) :1027-8 (1988), Franks S., Journal of Endocrinology 129:317-319 (1991) and Watson et al, Journal of Endocrinology 129 (Suppl.) Abstract No. 206 (1991)). In this study, affected women had significantly higher serum testosterone concentrations than non-affected subjects. There was no significant difference between men with MPB and those without, but the numbers in our study groups were small and androgens have been implicated in the cause of MPB. It has been suggested that, in women with PCO, there is abnormal regulation of the enzyme P450cl7α, which catalyses the rate limiting step in androgen biosynthesis in ovary and adrenal (Rosenfield et al , Fertil . Steril . 53(5):785-91 (1990) and Barnes et al , N. Engl . J. Med. 320(9) :559-65 (1989) ) . The idea that such an abnormality of androgen biosynthesis/metabolism is causative in PCO and premature MPB is very attractive, as it provides a unifying hypothesis which may explain apparent inconsistencies, such as the existence of PCO in more than 80% of women with congenital adrenal hyperplasia (Hague et al , Clin . Endocrinol . 33:501-10 (1990)). There are likely to be several other genes which modify the expression of PCO. For example, there is a genetic tendency for obesity and obese women with PCO are more likely to be symptomatic than their lean counterparts (Kiddy et al , Clin . Endocrinol . Oxf . 32(2) :213-20 (1990) ) . The effects of obesity in PCO seem to be mediated by insulin resistance or hyperinsulinaemia. Insulin resistance is known to be a feature of women with PCO regardless of body weight (Robinson et al , Clinical Endocrinology 36:537-543 (1992) and Dunaif et al, Diabetes 38:1165-1174 (1989)) . Furthermore the influence of obesity on symptoms can be reversed, at least partially, by calorie restriction. An obese, anovulatory woman with PCO may ovulate and go on to conceive in response to weight reduction alone (Kiddy et al , Clinical Endocrinology 36:105-111 (1992)). Similarly there are obvious racial genetic variations in the biology of androgen-dependent hair follicles. This will manifest itself in the presence and degree of hirsutism. However, in spite of secondary genetic effects, PCO can be regarded in all racial groups as an autosomal dominant disease, with virtually full penetrance. EXAMPLE 2 - Genetic Linkage Analysis The underlying discovery of the present invention is that polycystic ovary (PCO) is an autosomal dominantly inherited disorder with a male phenotype of male pattern baldness. The underlying biochemical abnormality may affect the regulation of the enzyme P450c 17α, which catalyses the rate limiting step in androgen biosynthesis in the ovary and adrenal. This enzyme is coded for by the gene CYP17. A YAC clone containing CYP17 has been isolated; its presence was confirmed by restriction mapping and sequencing of the PCR products from exons two and five. Purified YAC DNA was used as a template for Alu PCR, the products of which were labelled to map the human CYP17 gene to chromosome 10q24 - 25 by fluorescent in si tu hybridisation. Using the recent linkage map generated by Weissenbach et al , markers covering the region 10q23 - 26 were used to identify linkage with the disease locus. These markers are PCR based polymorphic loci containing short tracts of (AC)n repeats. The PCR for each marker were conducted in a total reaction volume of 25μl containing, 20ng of human DNA, 50pmol of each primer, CA BIO reaction buffer with lOOμM of each αP 32 -dATP/dCTP/dGTP/dTTP (Pharmacia) and 1 unit of Tag polymerase (Cambio) . The reaction was performed in an automated thermal cycler (Perkin-Elmer/Cetus) with 35 cycles of denaturing at 94°C (1 min) , annealing at 56°C (1 min) and extension of 72°C (1 min) . An initial denaturing step of 10 mins at 94°C was employed with a final extension at 72°C for 10 mins. Four microlitres of the PCR product were subsequently run on a 6% polyacrylamide sequencing gel electrophoresis at 65 watts. The gels were dried and exposed to KODAK X-OMAT ™ film for 36 hours. This procedure has allowed the typing of the alleies for a genotype of each individual member from ten of the pedigrees for the two markers D10S198 and D10S209. Based on the evidence that CYP17 maps to chromosome 10q24, linkage to this region has been investigated. Linkage analysis were carried out between the PCO locus and the marker loci using the LINKAGE 5.2 package, assuming an autosomal dominant mode of inheritance and a disease frequency of 1 in 10. With the a priori knowledge that the candidate gene, CYP17, maps to this interval, the generation of a pairwise lod score of z=1.38, 5=0.11 between the PCO locus and D10S198 is highly suggestive of the presence of the gene in this region. Multipoint analysis would support a location proximal to D10S198. EXAMPLE 3 Identification of a Polymorphism and PCR-based Test for Affected Individuals In affected individuals, a polymorphism, in which a T is replaced by a C, was found to exist within the 5' transcribed region of the cypl7 gene at -34bp from the printed sequence (Picado-Lenard S. Miller, DNA 6(5) 439- 448 (1987) ) . This base change creates a recognition site for the restriction enzyme MspKL (NEB) . A PCR fragment of 459bps containing the polymorphism is generated using the following primers: Forward CATTCGCACTCTGGAGTC (SEQ.ID.l) Reverse AGGCTCTTGGGGTACTTG (SEQ.ID.2) Polymerase chain reaction (PCR) amplification of genomic DNA was performed using AMPLITAQ ™ DNA polymerase (Cambio) and a PE KIN ELMER-CETUS ™ thermocycler. The 25μl amplification mixture contained 25ng of genomic DNA, 50pmoles of each primer, lx CAMBIO reaction buffer, with lOOμM each of dTTP/dCTP/dGTP/dATP and 1 unit of Taq Polymerase (Cetus) . The reaction was performed with 35 cycles of denaturation at 95°C (1 min) , annealing at 56°C (1 min) and extension at 72°C (1 min) . An initial denaturation step of 5 minutes at 95°C was employed with a final extension at 72°C for 10 minutes. An enzyme quantity of 0.2-0.5 units per reaction with 3μls buffer (+BSA) were added directly to the PCR mixture to a final volume of 30μls and incubated overnight at 37°C. To each sample, 5μls of 6x gel loading buffer (Sambrook et al . , "Molecular Cloning - A Laboratory Manual", Cold Spring Harbor Laboratory Press, (2nd Edition) 1989) were added and 20μls of this were loaded onto a 6% polyacrylamide non-denaturing gel (Severn Biotech) . The fragments were then separated by electrophoresis. The gel was subsequently stained with ethidium bromide (lOOμls of lOmg/ml Sigma) in lxTBE (0.9M tris, 0.9M boric acid, 0.5M EDTA) for 30 minutes and then destained in lxTBE for 10 minutes. If the base change is present in both alleies (homozygote affected individuals) fragments will be generated of 124 and 335bp. Heterozygous individuals for this mutation will have all three fragments present; 459, 335 and 124bp. A homozygous unaffected individual would only demonstrate the uncut product of 459bp. EXAMPLE 4 Association Studies based on the Mutation The incidence of the mutation in the groups is as follows: Consecutive probands with PCO is 74% (37 of 50) PCO family members 71% (96 of 136) Consecutive normal women 41% (7 of 17) Random DNA samples 46% (54 of 117) Hence using χ 2 analysis and comparing the groups:- 1. Consecutive PCO versus Consecutive normal: the mutation is associated with PCO group (p = 0.03) . 2. Consecutive PCO versus Random DNA samples: the mutation is associated with the PCO group (p < 0.002) . 3. All affected family members, as determined by hard evidence (ultrasound scan for PCO in women, photographic evidence for MPB in men) versus all normal family members, again as determined by hard evidence: 66 out of 88 (75%) of the affected family members have the mutation, whereas only 24 out of 48 (50%) of the normal family members have the mutation; the mutation is associated with the affected group (p = 0.01). All these groups were from women of Caucasian background and match for age. The conclusion is that the mutation is a risk factor for PCO/MPB. EXAMPLE 5 Genetic Segregation Analysis A two point analysis shows that five families generate a positive lod score of 1.8, twelve families generate a negative lod score of -2.65 (ie, exclusion) and five are uninformative. Within the five families generating the positive lod score, the mutation segregation is for the most part consistent. This suggests: (1) the mutation in CYP17 is associated with PCO/MPB; (2) for a number of families, this mutation may be causative; and (3) for the other families the mutation is probably inter-relating with one or possibly two other genes modulating the phenotype. Looking at the association of the presence/absence of the mutation with testosterone and androstenedione levels: (1) in men, the presence of the mutation significantly increases androgen levels for (a) all men combined, (b) affected men and (c) normal men, as determined by an unpaired t-test (p < 0.05) ; (2) in women with PCO who are ovular (regular cyclers) , ie who have no hyperinsulinaemia, the androgen levels are increased but the group is too small for significance; and (3) in both the normal individuals from the families and the anovular women from the families with PCO, the absence of the gene is associated with elevated androgens. This may well be due to the action of insulin in these groups, in which androgen levels are all significantly higher than the women with PCO and regular cycles, ie who are ovular. It therefore appears that PCO/MPB may be caused by CYP17 mutations alone in some families or individuals, by the effect of the mutation causing an up-regulation in the rate limiting step in androgen biosynthesis. It is likely that one or two other genes may be causally associated with PCO/MPB. These other genes may be involved in androgen synthesis or may affect insulin levels or action. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Imperial College of Science, Technology & Medicine (not US) (B) STREET: Sherfield Building, Exhibition Road, (C) CITY: London (E) COUNTRY: GB (F) POSTAL CODE (ZIP) : SW7 2AZ (A) NAME: CAREY, Adam Henry (US only) (B) STREET: St Mary's Hospital Medical School, Norfolk Place (C) CITY: London (E) COUNTRY: GB (F) POSTAL CODE (ZIP) : W2 1PG (A) NAME: FRANKS, Stephen (US only) (B) STREET: St Mary's Hospital Medical School, Norfolk Place (C) CITY: London (E) COUNTRY: GB (F) POSTAL CODE (ZIP) : W2 1PG (A) NAME: WILLIAMSON, Robert (US only) (B) STREET: St Mary's Hospital Medical School, Norfolk Place (C) CITY: London (E) COUNTRY: GB (F) POSTAL CODE (ZIP) : W2 1PG (ii) TITLE OF INVENTION: Test and Model for Polycystic Ovary Syndrome and Male Pattern Baldness (iii) NUMBER OF SEQUENCES: 2 (iv) COMPUTER READABLE FORM: 3.5 inch floppy disk, IBM compatible (low density) (v) CURRENT APPLICATION DATA: APPLICATION NUMBER: WO PCT/GB94/ (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: CATTCGCACT CTGGAGTC 18 (2) INFORMATION FOR SEQ ID NO: : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: AGGCTCTTGG GGTACTTG 18

Claims: CLAIMS 1. An isolated or recombinant DNA molecule including a mutant CYP17 allele which is associated with polycystic ovary syndrome (PCO) and/or male pattern baldness (MPB) . 2. A DNA molecule as claimed in claim 1, wherein the mutation is in the promoter region of the CYP17 gene. 3. A DNA molecule as claimed in claim 2, wherein T is replaced by C at position -34 from the translation initiation site. 4. An isolated or recombinant DNA molecule including a mutant CYP17 allele having a T→C mutation at position -34. 5. An isolated or recombinant DNA molecule including a mutant CYP17 promoter operatively coupled to a reporter gene to which it is not naturally coupled. 6. Recombinant DNA as claimed in any one of claims 1 to 5, which is in the form of a vector. 7. A host cell transformed or transfected with a vector as claimed in claim 6. 8. Recombinant or isolated DNA as claimed in any one of claims 1 to 5 which is in the form of a construct suitable for generating a transgenic animal. 9. A transgenic, non-human animal whose germline includes a transgene construct as claimed in claim 8. 10. The use of a host cell as claimed in claim 7 or an animal as claimed in claim 9 in a screen for compounds potentially useful in the treatment or prophylaxis of PCO and/or MPB. 11. A method of genetic analysis comprising detecting in a subject's DNA the presence of a CYP17 allele. 12. A method as claimed in claim 11, which is a method of determining the genetic predisposition of the subject to PCO and/or MPB. 13. An oligo- or poly-nucleotide specifically hybridising to a mutant CYP17 allele having a T→C mutation at position -34. 14. The use of a normal CYP17 gene or promoter in the preparation of a gene implant for the treatment or prophylaxis of PCO and/or MPB. 15. The use of a modulator of the production of P450cl7 by a mutant CYP17, or the activity of P450cl7 so produced, in the preparation of an agent for the treatment or prophylaxis of PCO and/or MPB. 16. The use as claimed in claim 15, wherein the modulator comprises a specific antisense polynucleotide complementary to all or part of a variant CYP17 gene.

Inventors: CAREY ADAM HENRY, FRANKS STEPHEN, WILLIAMSON ROBERT, CAREY, ADAM, HENRY, FRANKS, STEPHEN, WILLIAMSON, ROBERT
Applicants: CAREY ADAM HENRY, FRANKS STEPHEN, IMPERIAL COLLEGE, WILLIAMSON ROBERT, CAREY, ADAM, HENRY, FRANKS, STEPHEN, IMPERIAL COLLEGE OF SCIENCE, TECHNOLOGY AND MEDICI, WILLIAMSON, ROBERT
Application year: 1994
Main classification: C12N15
IPC codes: A61K48, A61K49, C07H21, C12N5, C12N9, C12N15, C12Q1